Some hormones, neurotransmitters and bacterial toxins affect cellular metabolism by altering regulatory pathways involved in the generation of second messengers. In some instances, binding of ligand to a receptor site on the exterior of the cell surface triggers an effect on an intracellular target. Coupling of receptor to effector is mediated through guanine nucleotide-binding (G) proteins. The activities of some of these G proteins are altered by certain bacterial toxins (e.g., pertussis toxin, cholera toxin) through a covalent modification known as ADP-ribosylation. Cholera toxin-catalyzed ADP-ribosylation results in activation of the G protein and persistent stimulation of the effector. The toxin-catalyzed reaction is in turn enhanced by 20 kDa guanine nucleotide-binding proteins, termed ADP-ribosylation factors or ARFs. In prior studies, cDNA clones for three different ARFs were isolated from human and bovine libraries; based on hybridization patterns of mammalian poly(A)+ RNA with a bovine ARF 2 probe, it was postulated that other ARF genes existed. Using differential hybridization with specific cDNA and oligonucleotide probes, cDNA clones that encoded new ARF forms were isolated from a cyclic AMP-differentiated HL-60 Lambda ZAP library. Thus far, six different putative ARF proteins have been described; they would appear to fall into three classes based on size (175, 180, or 181 amino acids), amino acid identity, and regions of amino acid sequence homology.